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Results for “target validation”

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PubMed2005

Biology calls the targets: combining RNAi and disease biology.

Target-based drug discovery starts with the identification of target genes and their respective protein products (associated with or controlling a disease-relevant phenotype) that, when inhibited or activated, ameliorate the associated disease. To identify disease-relevant genes, robust tools are needed to allow biology-driven target discovery and validation. Moreover, insight into the underlying biology of a disease is essential to model a disease in vitro. Key questions are: What are the disease hallmarks? What are, from a biological point of view, the best points for therapeutic intervention? How can scientists model these points in vitro? What is the desired target profile? The closer the cellular models resemble the disease situation, the better the target profile will be. The profile is the set of biological data needed to accept the target for drug discovery. In this review, a focused approach for target discovery and validation is presented. Arrayed adenoviral siRNA libraries and disease-based cellular models are used that generate high-quality and functionally validated targets.

Animals↗

PubMed2005

Targets for human encoded microRNAs in HIV genes.

MicroRNAs (miRNAs) are increasingly being shown to play vital roles in development, apoptosis, and oncogenesis by interfering with gene expression at the post-transcriptional level. miRNAs, in principle, can contribute to the repertoire of host pathogen interactions during HIV-1 infection. Using a consensus scoring approach, high scoring miRNA-target pairs were selected which were identified by four well-established target prediction softwares. While hsa-mir-29a and 29b target the nef gene, hsa-mir-149 targets the vpr gene, hsa-mir-378 targets env, and hsa-mir-324-5p targets the vif gene. Not only were the minimum free energy values lowest for the bound complex, but also, the rules so far observed for microRNA-target pairing, viz., a continuous stretch of 6-7 base pairing towards the 5' end of the miRNA and incomplete complementarity with the target sequence, were found to be valid. The target regions were highly conserved across the various clades of HIV-1. microRNA expression profiles from previously reported microarray based experiments show that the five human miRNAs are expressed in T-cells, the normal site of infection of HIV-1 virus. This is the first report of human microRNAs which can target crucial HIV-1 genes including the nef gene, which plays an important role in delayed disease progression.

Base Pairing↗

PubMed2004

Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain.

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis ) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14+/-0.013 mM and 0.19+/-0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a K(i) of 2.1+/-0.9 nM. A truncated BC domain that included amino acids 2-560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1+/-0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

Acetyl Coenzyme A↗

PubMed2004

RNA interference: from model organisms towards therapy for neural and neuromuscular disorders.

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology and we address problems to be solved in developing RNAi-based therapies.

Animals↗

PubMed2001

[Comorbidity indexes: review of the literature and application to studies of elderly population].

BACKGROUND: Older patients often suffer concomitant chronic diseases in addition to various health disorders. When attempting to study one of these diseases, it is necessary to consider the complexity of the patient's health status. Comorbidity is a cause of confusion, especially for elderly patients with multiple diseases. Comorbidity indexes have been developed in order to measure this complexity. METHODS: We focused this article on a review of the literature in order to identify and examine the validity of comorbidity indexes applied to the elderly population. RESULTS: Five indexes have been used to measure comorbidity in patient populations with complex health situations: the Cumulative Illness Rating Scale (CIRS), the Kaplan-Feinstein index, the Charlson index, the Index of Coexistent Disease (ICED), and the Total Illness Burden Index (TIBI). Each of these indexes included a series of domains which vary according to the authors'view of comorbidity. These indexes were validated under different conditions with similar targets. Their validation fields limited their use and extrapolation of results. Only the CIRS, the Charlson index, the Kaplan-Feinstein index and the ICED were validated and applied to old patients. However, the Charlson index was found to be limited in recording the entirety of the old patients'pathologies, and in patients with cognitive deficits, only CIRS appeared to be sufficiently trustworthy because it allows a comprehensive recording of all the comorbid disease from clinical examination and medical file data. CONCLUSION: When studying chronic disease states in frail elderly patients, it is essential to consider comorbidity using standard validated indexes in order to get a comprehensive assessment of the patient's situation and avoid neglecting diseases and handicaps. Further studies are necessary to develop and validate tools specifically adapted to the elderly population.

Activities of Daily Living↗

PubMed2001

[Tissue array technology for translational research. From gene discovery to application].

Large scale scanning of the human genome has become possible with the introduction of DNA microarray. The ability to survey the expression of up to 5000 to 50,000 genes in a single experiment provides significant new opportunities, as well as new challenge. It will be important to translate genomic scale information on cancer biology to functional or clinical application. This requires prioritization of hundreds of targets discovered, functional validation of these targets, as well as a thorough knowledge of the involvement of the candidate target genes in vivo in human tissue. We have developed a tissue array technology for genome scale expressional and clinical cancer research. This technology enables high-throughput molecular analysis of large number of specimens. Our tissue arrays are constructed by arranging the cylindrical biopsies of 2.0 mm diameter from 60 individual tumor tissues into a tissue array block, which is then sliced into 200 or more identical slides for probing RNA or protein targets. A single immunohistochemistry or in situ hybridization experiment provides information on all 60 specimens on the slides, while subsequent sections can be analyzed with other probes or antibodies. We produced gastric cancer tissue array slides with various kinds of subsets, including 600 subsequent cancer cases, 100 preneoplastic lesions, 60 metastatic lesions, 60 synchronous cancers, 60 metachronous cancers, 60 young age patients, and 120 familial cases. We searched the presence of Epstein-Barr virus in those cancer specimens. We also applied 10 antibodies in those samples and stratify the prognostic significance of these antibodies. Tissue array technology expand the scope of high-throughput molecular analysis of archival tissue specimens with multiple probes for specific genes or proteins for functional or clinical application.

Antibodies↗

PubMed2005

[The application of RNAi to the medical genetics].

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules have raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology.

Gene Silencing↗

PubMed2001

A review of the evidence for a disengage deficit following parietal lobe damage.

We review the literature on response times to ipsilesional and contralesional targets following spatial precues in patients with damage involving the left- and right-parietal lobes with the aim of appraising the 'disengage deficit' reported initially by Posner and colleagues (Posner MI, Cohen A, Rafal RD. Neural systems control of spatial orienting. Proceedings of the Royal Society of London, B 1982;298:187-98). The data of individual subjects from a sub-sample of studies were submitted to analyses of variance, and data from all studies meeting our selection criteria were submitted to meta-analytic procedures (Hunter JE, Schmidt FL. Methods of meta-analysis: correcting error and bias in research. Newberg Park: Sagge Publications, 1990). Findings from both types of analysis conducted on data from patients with right-hemisphere lesions indicate that: (1) the disengage deficit phenomenon is robust following peripheral cues, but not following central cues; (2) the disengage deficit is large at shorter cue-target stimulus onset asynchronies (SOAs), and decreases as SOA increases; (3) the disengage deficit is larger in patients with a diagnosis of hemispatial neglect; and (4) although the magnitude of the disengage deficit appears to increase with increases in lesion size, multilobar vs unilobar involvement did not significantly alter the pattern of the disengage deficit. We also show that responses to validly cued targets in the contralesional hemispace were significantly slower than for validly cued targets in ipsilesional hemispace. Similar, but usually smaller, effects were observed in patients with homologous left-hemisphere damage. The implications of these results for current models of the role of the parietal lobes in attentional orienting are discussed.

Animals↗

PubMed2005

Current status of malaria control.

Malaria caused by Plasmodium parasites kills approximately 1-3 million people and causes disease in 300-500 million people annually throughout the world. The current approaches to curtail this disease include vector control, vaccination, immunotherapy and chemotherapy. The vector control is achieved by reducing vector density, interrupting their life cycle, and creating a barrier between the human host and mosquitoes. A number of vaccine candidates are being clinically tried and R&D effort in this direction is coming in a big way. Currently there are only limited safe drugs for the treatment of this disease, however, reports of emerging resistance against existing drugs warrant the introduction of new drugs, which are unlikely to come from pharmaceutical industries because of limited commercial opportunities. One of the most important current approaches to develop new drugs involves the synthesis of chemical libraries and evaluate them against most validated biochemical targets of malarial parasite. Although a number of such targets in antimalarial drug development are known today, yet only validated and selective biochemical targets including mitochondrial transport, glycolic pathway, folate pathway, proteases and heme metabolism, apicoplast metabolism, glycophospatidyl inositol, lipid metabolism (glycerophospholipids), peptidyl deformylase and oxidative stress in parasite-infected erythrocytes have been discussed here. The well known antimalarial drugs and different drug combinations for the treatment of malaria are also briefly reviewed. A survey of the recently discovered new molecules active against malaria has also been narrated. Lastly, the future of malaria chemotherapy and new directions emerging from literature has been elucidated.

Animals↗

PubMed2001

Exploiting genomics to discover new antibiotics.

There is an urgent need to develop new classes of antibiotics to tackle the increase in resistance in many common bacterial pathogens. One strategy to develop new antibiotics is to identify and exploit new molecular targets and this strategy is being driven by the wealth of new genome sequence information now available. Additionally, new technologies have been developed to validate new antibacterial targets, for example, new technologies have been developed to enable rapid determination of whether a gene is essential and to assess the transcription status of a putative target during infection. As a result, many novel validated targets have now been identified and for some, appropriate high-throughput screens against diverse compound collections have been carried out. Novel antibiotic leads are emerging from these genomics-derived targeted screens and the challenge now is to optimize and develop these leads to become part of the next generation of antibiotics.

Anti-Bacterial Agents↗

PubMed2003

Discovery and validation of new molecular targets for ovarian cancer.

The occult progression of tumors within the peritoneal cavity results in the late diagnosis and high mortality rate of ovarian cancer. An improved understanding of the molecular biology of ovarian cancer has led to the discovery of novel molecules as targets for the treatment of ovarian cancer. This review highlights the latest advance of mucins, which are upregulated in cancer cells and interact with the epidermal growth factor receptor family to regulate cell behavior via signaling pathways associated with malignant transformation, invasion and metastasis. Disruption of these molecules might represent a novel therapeutic approach in treating ovarian cancer. We also describe the recent development and validation of the most studied mucins (MUC1 and MUC16), ErbB-2 (Her2/neu) and folate binding protein as target-based immunotherapy of ovarian cancer.

Animals↗

PubMed2001

Spatial attention triggered by eye gaze increases and speeds up early visual activity.

What are the neuronal correlates of reflexive shifts of attention triggered by eye gaze direction? Event related potentials (ERPs) were measured on 14 subjects performing a spatial attention task where eye gaze direction of a face cued the location of a forthcoming target. Subjects were faster in detecting a validly cued target, i.e. one appearing at the location the eye was gazing at, compared to invalidly cued targets, despite the non-predictive value of the eye cues. ERP results showed an enhanced and earlier occipito-parietal P1 and N1 for valid trials, demonstrating the early modulation of visual input by attentional allocation. These findings provide the first evidence that social attention can rapidly modify the processing of visual information in extrastriate cortex.

Adult↗

PubMed1979

Social validation: the evolution of standards of competency for target behaviors.

The use of social validation procedures has become widespread in recent years. Although most researchers have used social validation procedures to select target behaviors and to evaluate whether the changes produced by a treatment program should be considered socially useful, little attention has been focused upon using the social validation process to determine the optimal levels for target behaviors. This paper suggests several ways in which social validation procedures can be employed in order to select when and how much to change target behaviors.

Behavior Therapy↗

PubMed2001

Development of antimicrobial agents in the era of new and reemerging infectious diseases and increasing antibiotic resistance.

During the past 2 decades, new infectious diseases have appeared and old ones previously thought to be controlled have reemerged. New and reemerging infectious agents will continue to pose serious threats well into the 21st century. The prediction that the threat of infectious disease may not diminish is supported by evidence that infectious agents cause many chronic diseases and cancer of previous unknown etiology. Moreover, the utility of existing antimicrobial agents is rapidly eroding, tipping the balance in favor of multidrug-resistant pathogens, and there appear to be few, if any, new classes of drugs currently in clinical development. The need for research directed toward development of new antibiotics has never been greater. Advances in research technologies and microbial genome sequencing in the past decade have led to identification of a large number of new targets. Functional genomics and integrative biology should validate these targets and provide the best opportunity for developing effective new therapies, improved diagnostic techniques, and better tools to understand host-pathogen interactions.

Animals↗

PubMed2000

Chemical ligands, genomics and drug discovery.

The sequencing of the human genome and numerous pathogen genomes has resulted in an explosion of potential drug targets. These targets represent both an unprecedented opportunity and a technological challenge for the pharmaceutical industry. A new strategy is required to initiate small-molecule drug discovery with sets of incompletely characterized, disease-associated proteins. One such strategy is the early application of combinatorial chemistry and other technologies to the discovery of bioactive small-molecule ligands that act on candidate drug targets. Therapeutically active ligands serve to concurrently validate a target and provide lead structures for downstream drug development, thereby accelerating the drug discovery process.

Journal Article↗

PubMed2006

Her-2 targeted therapy: beyond breast cancer and trastuzumab.

Her-2 is a validated therapeutic target in breast cancer. The two critical questions that remain regarding Her-2 targeting concern 1) the relevance of Her-2 inhibition in other malignancies and 2) the ability of novel agents to achieve greater Her-2 inhibition than trastuzumab. The contribution of cell signaling effects and immunologic mechanisms to the effect of trastuzumab in vivo remains poorly understood. Thus, the preclinical data that support the greater efficacy of novel Her-2 antibodies or small molecule tyrosine kinase inhibitors remain to be validated in clinical trials. In this review, we discuss the evidence from recent trastuzumab clinical trials as a point of departure for consideration of novel Her-2 targeted therapies. Preliminary results from early clinical trials suggest that Her-2 tyrosine kinase inhibitors may extend the population for which this strategy offers therapeutic effect.

Antibodies, Monoclonal↗

PubMed2022

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket.

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis in vivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose in vivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

Biological Products↗

PubMed2004

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis.

We developed a versatile, high-throughput genetic screening strategy by coupling gene mutagenesis and expression profiling technologies. Using a retroviral gene-trap vector optimized for efficient mutagenesis and cloning, we randomly disrupted genes in mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that transcriptional target genes of platelet-derived growth factor (PDGF) can be efficiently and reliably identified in physiologically relevant cells and are immediately accessible to genetic studies to determine their in vivo roles and relative contributions to PDGF-regulated developmental processes. The same platform can be used to search for genes of specific biological relevance in a broad array of experimental settings, providing a fast track from gene identification to functional validation.

Animals↗